Elastic anisotropy of bivalve hinge-ligament.

Compressive stress-strain properties of an elastic ligament of a bivalve, Pseudocardium sachalinensis were investigated in the swollen state in water. The ligament is a calcified tissue, composed of calcium carbonate and insoluble protein which is rich in methionine S-oxide residue [Kikuchi, Y. and Tamiya, N., J. Biochem. (Tokyo) 89, 1975-1976 (1981)]. X-ray diffraction study showed that calcium carbonate existed only in orthorhombic aragonite form, and that all the crystal c-axes of the unit cell orientate nearly in the growing direction of the ligament. The uniaxial compression modulus for the growing direction was appreciably larger than those for the other two directions, while the anisotropy of the modulus was absent for a decalcified ligament. Thus the mechanical anisotropy of the ligament could be explained by means of the uniaxially oriented structure of aragonite crystals being dispersed in a nearly isotropic protein matrix.

Hereditary amyloidosis: evidence against early amyloid deposition.

Twelve members of the Indiana hereditary amyloidosis type II kindred were tested for the presence of amyloid deposits. All were young adults (age 26-37), with no evidence of disease and with 1 affected parent. Six were found to be carriers of the variant gene, by DNA testing and/or reduced serum retinol-binding protein levels. Nevertheless, no amyloid could be found in any skin, rectal, or carpal tunnel biopsy specimens. Our results suggest that hereditary amyloidosis type II is a true late-onset disease, in which accumulation of amyloid does not start until late in life--perhaps only a short time before symptoms appear.

Developmental regulation of the mRNAs for elastins a, b and c in foetal-calf nuchal ligament and aorta.

The data presented clearly suggest that relative amounts of mRNAs for elastins a, b and c are developmentally regulated in foetal-calf nuchal ligament and aorta and that this regulation is tissue-specific. In nuchal ligament, at earlier stages of foetal development, the relative amounts of mRNAs for elastins a and b are very low. After the foetal age of about 6 months the relative amount of mRNA for elastin b begins to increase. This is followed by an increase in the relative amount of mRNA for elastin a. In aorta, with increasing foetal age, the relative amounts of mRNAs for elastins b and c increase and decrease alternately. The relative amounts of mRNA for elastin a remain low, with only marginal increases with foetal age. A possible self-aggregation role of elastin a in elastogenesis is proposed.

Amyloid localized to tenosynovium at carpal tunnel release. Natural history of 124 cases.

One hundred fifty-two patients with amyloid in the tenosynovium who had carpal tunnel release were identified. Twenty-eight patients were excluded because of systemic amyloidosis: primary systemic amyloidosis (AL) in 24, secondary amyloidosis (AA) in 3, and familial amyloidosis (AF) in 1. The remaining 124 patients (82%) had carpal tunnel syndrome with local deposition of amyloid and no evidence of systemic amyloidosis. Median survival of the 124 patients from diagnosis of amyloidosis was 12 years. Only two patients had systemic amyloidosis develop--9 and 10 years after recognition of tenosynovial amyloid. Of particular interest were 12 patients who had an M-protein in the serum or urine. None of the 12 patients have had evidence of systemic amyloidosis or multiple myeloma during the median follow-up of 14 years. The authors conclude that amyloid may be localized to the tenosynovium and that systemic amyloidosis rarely develops during long-term follow-up.

Collagen types I and III and fibronectin in healing anterior cruciate ligament after reconstruction with carbon fibre.

The presence of the connective tissue components fibronectin and collagen types I and III have been demonstrated by immunohistological methods after reconstruction of the anterior cruciate ligament of pigs with carbon fibre. The carbon fibre ligament prosthesis was covered with pediculated fascia lata. During the initial 16-week period, fibronectin was seen both in the surrounding fascial layer and between the carbon fibres of the substitute. Type III collagen was also found, especially in the fascial layer, but collagen was absent from the carbon fibre prosthesis and was seen only in the fascial layer. Results indicate that the tissue in the 'neoligament' after carbon fibre reconstruction consists mostly of granulation tissue with a high amount of fibronectin, and type III collagen from mesenchymal cells without sufficient amount of type I collagen with tensile strength to withstand subsequent mobilization and rehabilitation.

The protein components of the 12-nanometer microfibrils of elastic and nonelastic tissues.

A procedure has been developed which is much more specific for the solubilization of the elastin-associated microfibrils from fetal bovine nuchal ligament using treatment with reductive saline in place of reductive guanidine hydrochloride buffer. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reductive saline extracts were shown to contain only five major protein bands with Mrs of 340,000, 78,000, 70,000, 31,000, and 25,000. The 31-kDa species was identified immunologically as the previously described macromolecule named microfibril-associated glycoprotein (MAGP) (Gibson, M. A., Hughes, J. L., Fanning, J. C., and Cleary, E. G. (1986) J. Biol. Chem. 261, 11429-11436). The proteins were purified by gel permeation, ion exchange, and affinity chromatography. Amino acid analyses showed that each protein had a profile which was distinct from that of MAGP although each was also high in acidic amino acids and cystine. The 340- and 78-kDa species were each demonstrated by immunoelectron microscopy with affinity-purified antibodies to be derived from the elastin-associated microfibris, and these were provisionally named microfibrillar protein 340 (MP340) and microfibrillar protein 78 (MP78), respectively. Each of the above antibodies gave a tissue distribution identical to that of anti-MAGP antibodies, and thus MP340 and MP78 also were identified with the 12-nm microfibrils of nonelastic tissues. MP340 was shown to absorb out completely the microfibrillar immunoreactivity of anti-(reductive guanidine hydrochloride extract) antibodies, indicating that MP340 was (a) the major microfibrillar constituent in these extracts and (b) the second unidentified microfibrillar antigen described previously. The relationship of the 70- and 25-kDa proteins to microfibrils is yet to be established. Immunoblot and immunoabsorption studies showed that MAGP and MP78 were immunologically related to MP340 but not to each other. Cyanogen bromide peptide mapping indicated that MAGP was structurally related to MP340. It is postulated that MAGP and MP78 are constituents of MP340 which in turn is the subunit of which the 12-nm microfibrils are composed.

Distribution of 35S-sulfate within the transseptal ligament of the mouse.

The distribution of 35S-sulfate-labeled macromolecules was examined within three regions of the transseptal ligament: the 1) mesial, 2) middle and 3) distal thirds. Swiss mice, 6 weeks of age, were injected with 35S-sulfate and killed after 1, 6, and 12 hours and 1, 2, 3, 4, 5, and 7 days. Silver grains and cell nuclei were counted on autoradiographs which had been counterstained by the Van Gieson method, and mean counts were analyzed statistically. Analysis of variance revealed no significant differences in mean number of cell nuclei between regions throughout the course of the experiment. 35S-sulfate was rapidly incorporated into the transseptal ligament macromolecules. Grain counts were highest 6 hours after injections: counts were highest over the middle and lowest over the mesial thirds of the ligament. The rate of grain removal was significantly higher in the middle third compared to the mesial or distal thirds (P less than 0.001) and was significantly lower in the mesial third compared to the middle or distal thirds (P less than 0.001). The half-life of labeled macromolecules was significantly greater in the mesial and distal thirds than in the middle third (P less than 0.005). The data demonstrate significantly higher rates of turnover of 35S-sulfate-labeled macromolecules in the middle region of the transseptal ligament. Since cellular density was similar throughout the transseptal ligament, higher turnover rates of 35S-sulfate-labeled macromolecules probably indicate higher rates of cellular activity in this region, possibly a result of tissue remodeling coincident to stresses generated by occlusal forces and physiologic drift of the adjacent teeth.

A reevaluation of the distribution of 3H-proline in the transseptal ligament of the mouse in radioautography.

It is generally accepted that there is uniform collagen metabolism within the periodontal and transseptal ligaments. The present study suggested regional variations in the incorporation and removal of 3H-proline within the transseptal ligament in radioautography, suggesting variable rates of collagenous protein remodeling coincident with physiological tooth movements. Highest numbers of silver grains were over the middle third of the ligament during both incorporation and removal phases (p less than 0.001). Rates of grain removal were greater in the middle than in mesial or distal thirds (p less than 0.001). The half-life of labeled proteins was significantly less in the middle than in mesial or distal thirds (p less than 0.005). Because there were no significant regional differences in cell numbers, regional variability in grain incorporation and removal within the transseptal ligament likely indicates regional differences in cellular synthetic or degradative activity coincident with remodeling of the transseptal ligament during physiological drift and suggests that the center of this ligament may experience more stress and, thus, remodels more rapidly.

Post-embedding methods for immunolocalization of elastin and related components in tissues.

Elastic tissue is composed of amorphous-appearing elastin and 12-nm diameter microfibrils, one component of which has recently been isolated and characterized as the 31 KD microfibril-associated glycoprotein MAGP. Monospecific antibodies to each of these components have been developed in this laboratory. The parameters that determine optimal localization of colloidal gold probes for post-embedding immunolabeling of elastic tissue components have been systematically studied in a variety of normal and developing tissues in mammals and birds. Protein A-gold probes stabilized with dextran have been shown to provide complexes that remain stable after more than 2 years. Conditions have been defined that permit precise localization within the extracellular matrix of antibodies to MAGP and to elastin, singly and together. Best results were obtained with acrylic resins (Lowicryl K4M or LR White). Fixation in glutaraldehyde or other aldehydic fixatives, with or without osmium, did not affect the immunostaining of elastic tissue with affinity-purified antibodies to tropoelastin, or to anti-[alpha-elastin] or anti-[alkali-insoluble elastin]. Immunostaining with the anti-MAGP antibody was less robust and was possible in tissues which had been fixed only lightly before embedding in Lowicryl K4M or LR White. This staining was enhanced by metaperiodate oxidation of the sections as well as by reduction of the tissues with sodium borohydride en bloc, followed by hyaluronidase digestion of the sections. The effects on immunostaining of a range of enzyme digestions have also been examined. Conditions have thus been defined that make possible detailed study of the relationship between elastic tissue, elastin-associated microfibrils, and other microfibrillar structures in normal and abnormal tissues during development and aging.

The diaphragm: anatomic, pathologic, and radiologic considerations.

The diaphragm, by virtue of its complex anatomy and multiple ligamentous connections to both thoracic and abdominal structures, is more than a simple partition between the chest and abdomen. Cross sectional images of the diaphragm and peridiaphragmatic processes can be confusing unless the radiologist is aware of the normal structure of the diaphragm, its attachments to the body wall, and the multiple ligaments that attach to the diaphragm.

Replacement of the anterior cruciate ligament with a bone-ligament-bone anterior cruciate ligament allograft in dogs.

Acute replacement of the canine anterior cruciate ligament (ACL) with a frozen, bone-ligament-bone anterior cruciate ligament preparation was studied using biochemical, immunologic, and biomechanical testing methods. Nine dogs were used for the study, six dogs received allografts and three received autografts. No tissue antigen matching was performed. All nine dogs were killed nine months after surgery. Necropsy examination revealed that the ACL was not present in three joints (one autograft, two allografts). The two autograft and four allograft ligaments available for mechanical testing sustained mean maximum loads that were 10% and 14%, respectively, of the mean maximum loads sustained by the contralateral ACL. Autoradiography indicated that cellular activity was more pronounced in the autograft specimens. Hydroxyproline uptake was 200% and 45% of normal in the autograft and allograft ligaments, respectively. Both autograft and allograft specimens were producing Type I collagen at the time of killing. Antidonor dog leukocyte antigen (DLA) antibody was detected in the synovial fluid taken at the time of killing from six of six dogs that received allografts and in zero of three dogs that received autografts.

[A histochemical study of acid glycosaminoglycans on normal and pathological cartilage, ligaments and several other connective tissues].

Histochemical estimation of acid glycosaminoglycan was critically re-evaluated, using the meniscus, intervertebral disc, ossified yellow ligament, ganglion, Dupuytren's fascia and several other tissues. Each tissue was stained with toluidine blue, alcian blue, high iron diamine, low iron diamine, aldehyde fuchsin and dialyzed iron-ferrocyanide. Digestion techniques for GAG were used in these staining methods, and the effects of protease inhibitors (PI) on digestion were also examined. In this study, the optimal temperature for digestion with Streptomyces hyaluronidase was between 37 degrees C and 43 degrees C, which varied according to the tissue examined. The addition of PI seemed necessary because the enzymatic treatment without PI resulted in an excessive decrease of staining. Protease-free chondroitinase ABC, which did not excessively decrease staining results, was found to be more useful than chondroitinase ABC without PI.

Desmosine and isodesmosine as cross-links in the hinge-ligament protein of bivalves. 3,3'-Methylenebistyrosine as an artefact.

Desmosine and isodesmosine were detected in an invertebrate molluscan species, i.e. in an insoluble protein in the hinge ligament of a bivalve species, Sakhalin surf clam (Pseudocardium sachalinensis, in family Mactridae). The protein is rich in glycine and methionine S-oxide but devoid of hydroxyproline and hydroxylysine. 3,3'-Methylenebistyrosine was also detected in the HCl hydrolysate of the hinge-ligament protein, but it was found to be an artefact produced from tyrosine and formaldehyde derived from methionine S-oxide during the HCl hydrolysis of the protein.

Chemical taxonomy of the hinge-ligament proteins of bivalves according to their amino acid compositions.

The proteins in the hinge ligaments of molluscan bivalves were subjected to chemotaxonomic studies according to their amino acid compositions. The hinge-ligament protein is a new class of structure proteins, and this is the first attempt to introduce chemical taxonomy into the systematics of bivalves. The hinge-ligament proteins from morphologically close species, namely mactra (superfamily Mactracea) or scallop (family Pectinidae) species, showed high intraspecific homology in their compositions. On the other hand, inconsistent results were obtained with two types of ligament proteins in pearl oyster species (genus Pinctada). The results of our chemotaxonomic analyses were sometimes in good agreement with the morphological classifications and sometimes inconsistent, implying a complicated phylogenetic relationship among the species.

Cervical myelopathy attributable to pseudogout. Case report with radiologic, histologic, and crystallographic observations.

A 77-year-old woman complained of numbness in her hands and feet, progressive unsteadiness, weakness, and loss of proprioception of six months' duration. A myelogram revealed stenosis of the spinal canal at the levels of C2-3, T6-7, L2-3, and L3-4. On computerized tomography scan, a large dorsal, epidural, soft tissue mass and focal calcification of the ligamentum flavum were seen at C3. Laboratory studies ruled out gout, collagen disease, vitamin B12 deficiency, syphilis, parathyroid, and thyroid disease. At decompressive laminectomy, a nodular mass in the ligamentum flavum (C2-4) was found and removed. Three months after operation, the neurologic symptoms had improved. Histologic examination of the elastic ligament revealed deposits of birefringent crystals, which were identified by X-ray diffraction as calcium pyrophosphate dihydrate (CCPD). Only about six cases of myelopathy attributable to deposits of CPPD appear to have been previously reported.

Isolation and characterization of a pentameric amino acid from elastin.

An unknown amino acid was purified from bovine ligament elastin. The compound was shown by proton NMR to have a pyridinium ring structure similar to isodesmosine, yet containing an olefinic double bond on one additional side chain which was not attached to the pyridinium ring. Mass spectral analysis confirmed the NMR data and indicated a parent compound with a mass of 653. A structure is proposed that is derived from the condensation of five lysine residues. The trivial name of pentasine is proposed for this compound.

Elastin in the human intervertebral disk. A histological and biochemical study comparing it with elastin in the human yellow ligament.

The elastic fiber and elastin in the human yellow ligament and intervertebral disk were studied histologically and biochemically. The elastic fiber in the human intervertebral disk, which until now had not been clearly identified microscopically, was observed clearly. We found the distribution of the elastic fiber in the intervertebral disk to be very sparse and irregular, and its diameter was small, being about one-tenth of that found in the yellow ligament. The elastin contents of the yellow ligament and intervertebral disk were 46.7% +/- 0.9% and 1.7% +/- 0.2% respectively (mean +/- SE) of the total dry weight. The amino acid composition of elastin in the yellow ligament is similar to that of other tissue, as reported in the literature; however, that found in the intervertebral disk is significantly different. It would appear, therefore, that the elastin in the intervertebral disk is of a different type from that found elsewhere.

Axial computed tomographic anatomy of the foot. Part II: Midfoot.

Osseous and soft tissue anatomy of the human midfoot is presented as visualized on high resolution axial computed tomography (CT). Because of the proven and potential capabilities of CT in the diagnosis of midfoot disorders, this report serves as an important standard for comparison with clinical studies.

In vitro mechanical properties of equine tendons in relation to cross-sectional area and collagen content.

The mechanical properties of the deep digital flexor tendon (DDFT), the superficial digital flexor tendon (SDFT) and the suspensory ligament (SL) of the hindlimb of the horse were studied in vitro. The tendons were observed at several morphologically distinct sites. The loaded tendon is homogeneously strained, in spite of large variations in cross-sectional area. Consequently the modulus of elasticity was inversely proportional to the corresponding cross-sectional area and ranged from 738 MPa (megaPascal, N mm-2) to 1398 MPa within the DDFT, from 1000 MPa to 1282 MPa within the SDFT and from 576 MPa to 669 MPa within the SL. The collagen content was inversely proportional to the cross-sectional area and proportional to the modulus of elasticity. This stresses the influence of tendon composition on the mechanical properties, and also demonstrates the difficulty in judging the strength of a particular tendon or site within a tendon from its cross-sectional area. The respective tendons ruptured at strains of 10.0 per cent (DDFT), 12.3 per cent (SDFT) and 11.0 per cent (SL). The influence of strain rate on the modulus of elasticity is small, and these tendons may therefore be considered as non-linear elastic structures. The average hysteresis is about 5 per cent.

CL glycoprotein is the tissue form of type VI collagen.

CL glycoprotein (CLGP), the 140,000-dalton collagenous glycoprotein, has been isolated from fetal bovine aorta and nuchal ligament, in milligram amounts in its reduced and alkylated form, using a multistage procedure. This material exhibited a characteristic amino acid composition with a consistent ratio of hydroxylysine to hydroxyproline (approximately 1:1). Digestion of CLGP with bacterial collagenase yielded three discrete noncollagenous fragments. Monospecific anti-CLGP antiserum exhibited strong cross-reactivity with the pepsin-resistant polypeptides of type VI collagen. CLGP was also prepared in the unreduced disulfide-bonded form and in a partially reduced form, using brief treatment with cysteine. On treatment with pepsin these preparations yielded resistant peptides corresponding in size to the longer and shorter forms, respectively, of type VI collagen. A slightly larger, soluble form of CLGP (Mr = 150,000) was detected in the media from cultures of aortic smooth muscle cells and nuchal ligament fibroblasts. The evidence indicates that CLGP is the native form in which type VI collagen is present in the tissues and that it consists of three structurally distinct polypeptide chains, each about 140 kDa in mass, which are disulfide bonded into a triple-helical molecule. The CLGP molecules appear to be present in the tissues as dimers and larger aggregates, stabilized by intermolecular disulfide bonding. The distribution of type VI collagen will thus be as described in our earlier immunofluorescence studies with anti-CLGP antiserum (Gibson, M.A., and Cleary, E.G. (1983) Collagen Relat. Res. 3, 469-488).